Differential Role of Glutamate Dehydrogenase in Nitrogen Metabolism ...

Plant Physiol. (1984) 76, 536-540 0032-0889/84/76/0536/05/$0 1.00/0

Differential Role of Glutamate Dehydrogenase in Nitrogen Metabolism of Maize Tissues' Received for publication March 27, 1984 and in revised form July 2, 1984

VICTOR MANUEL LOYOLA-VARGAS2 AND ESTELA SANCHEZ DE JIMENEZ* Departamento de Bioquimica Vegetal, Facultad de Quimica, Universidad Nacional Aut6noma de Mexico, 04510 Mexico, D.F., Mexico ABSTRACT Both call and plantlets of maize (Zea mays L. var Tuxpefio 1) were exposed to specific nitrogen sources, and the aminative (NADH) and deaminative (NAD+) glutamate dehydrogenase activities were measured at various periods of time in homogenates of calli, roots, and leaves. A differential effect of the nitrogen sources on the tissues tested was observed. In callus tissue, glutamate, ammonium, and urea inhibited glutamate dehydrogenase (GDH) activity. The amination and deamination reactions also showed different ratios of activity under different nitrogen sources. In roots, ammonium and glutamine produced an increase in GDH-NADH activity whereas the same metabolites were inhibitory of this activity in leaves. These data suggest the presence of isoenzymes or conformers of GDH, specific for each tissue, whose activities vary depending on the nutritional requirements of the tissue and the state of differentiation.

In recent years, the demonstration of glutamate synthase activity (GOGAT, EC 1.4.7.1.) in several plant systems (15, 26, 27) has lead to the idea that glutamate is synthesized from glutamine rather than from ammonium (12). However, the synthesis of glutamate from 2-oxoglutarate and ammonium by the enzyme GDH3 (EC 1.4.1.2.) has recently been reevaluated and it is considered as an important reaction in the assimilation of inorganic nitrogen in plants (18). Correlation between the activity of this enzyme and the formation of organic nitrogen compounds has been observed (25). In addition, several GDH isoenzymes are present in different plant tissues (7, 14, 28) and the pattern of isoenzymes varies depending on the source of nitrogen (10, 14), suggesting specific roles for each isoenzyme. Although there are several reports on the regulation of GDH by the nitrogen source (1, 8, 16), this information is derived primarily from C-3 plants (4, 9). The nitrogen source affects carbohydrate metabolism so markedly that the effects on regulation of GDH could be quite different in C4 plants. Therefore, we carried out studies on the regulation of GDH in tissue culture

of a C4 plant such as maize. Tissue culture has been used in biochemical studies because of its advantage over other models

in controlling the levels of nitrogen substrates (2, 5, 13, 29). However, there are very few reports using maize tissues (29). The present report is concerned with the effect of different nitrogen sources on the aminative and deaminative activities of GDH. A differential regulatory effect on both directions was observed which suggests that each isoenzyme or enzyme conformer has a specific role in cellular nitrogen metabolism. MATERIALS AND METHODS Plant Material. Corn plants (Zea mays L. var Tuxpefio 1) were grown in a green house for 17 d. At this stage, groups of 50 plantlets were sprayed with 150 ml of one of the following solutions: water (control), 20 mm NH4Cl, 40 mM KNO3, 20 mM NH4N03 plus 20 mM KNO3, 10 mM NH4 citrate, 10 mm glutamine, 10 mM L-glutamate, and 10 mM urea. Leaves and roots were harvested 3 d after this treatment and were processed for enzyme assays. Maize callus previously established in our laboratory from mature corn embryos of the same variety (21, 22) were grown in MS with 4 mg/l MCPP, 20 mg/l IAA, 0.01 mg/l kinetin, and 0.8% agar. For the metabolic experiments, 1 g of callus was subcultured in 2 X 12-cm tubes with 8 ml of the same medium but with different nitrogen salts as follows: 2, 20, or 40 mM NH4CI; 20 or 40 mM KN03; 20 mm NH4N03 plus 20 mM KNO3 (MS); 10 mm glutamate or 10 mM urea. Callus tissues were subcultured every 14 d and kept under darkness at 28C. Fifteen to thirty different samples were weighed to determine each point of the growth curves. Enzyme Extract. The tissues (callus, roots, or leaves), were homogenized with a polytron in 2.5 volumes (w/v) ofextraction buffer (50 mM Tris-HCl, 5 mm 2-mercaptoethanol, and 1 mm CaC12, pH 8.2, and 5% PVP). The homogenates were filtrated through four layers of muslin and centrifuged at 14,000g for 30 min. The supernatants were used for the enzyme determinations. The whole procedure was carried out at 4C. Enzyme Assay. The amination and deamination reactions of GDH activity were assayed using a Pye Unicam double beam spectrophotometer. The amination reaction was measured at 30C in 100 mm Tris-HCl, pH 8.2, containing 100 mm NH4Cl, 10 mm 2-oxoglutarate, and 0.16 mM NADH and 4 mM CaC12 and the 2-oxoglutarate-dependent oxidation of NADH followed at 340 nm. The deamination reaction was measured at 30C in 100 mm Tris-HCl, pH 9.4, containing 50 mM L-glutamate, 0.625 mM NAD+, and 1 mM CaCl2 by following the L-glutamatedependent reduction of NAD+ at 340 nm. Both reactions were initiated by addition of the enzyme extract. Enzyme units are expressed as nmoles ofcofactor oxidized (or reduced) per minute. Proteins. Peterson's modification of the Lowry method was used to measure proteins (17).

' Supported by CONACYT (National Council for Science and Technology Improvement) M6xico. Grant No. PCCBBNA-005076. Under the Academic Collaboration Program: Universidad Nacional Aut6noma de M6xico (UNAM)-Colegio de Postgraduados, Chapingo, M6xico. 2 Part ofthis work was carried out while V. M. L. V. was on sabbatical recess from Centro de Estudios Nucleares, UNAM. 3Abbreviations: GDH, glutamate dehydrogenase; MS, MurashigeSkoog medium; MCPP, 2-(2-methyl,4-chloro)phenoxypropionic acid. 536

ROLE OF GDH IN MAIZE

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FIG. 1. Maize callus growth curve in MS medium. (0), Fresh weight; (0), GDH-NADH activity; (U), GDH-NAD+ activity.

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FIG. 2. Effect of nitrogen source on the callus growth. Each value is the average of at least three independent observations. Weights were taken after 14 d of subculture. MS medium was used in all the cases with the specific nitrogen source. 1, 40 mm KNO3 + 20 mM NH4Cl; 2, 20 mM KNO3; 3, 40 mm KNO3; 4, 2 mM NH4Cl; 5, 20 mM NH4IC; 6, 40 mm NH4Cl; 7, 10 mm

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DAYS RESULTS GDH activity in both directions was followed through the growth cycle of the calli on MS medium. The activities did not show the same variation during this period (Fig. 1). While the GDH-NADH gave a main peak of activity about the 17th d (the initial stationary stage), GDH-NAD+ showed two peak of activ-

ity, one on the 9th d and the other on the 14th d of cultures (early and middle log phases, respectively). When different ammonium sources were studied, the growth of call was followed by measuring both fresh and dry weight through six subcultures. A similar pattern was obtained for dry weight. The best growth was observed in the MS medium (control) which has a balanced nitrogen source. In this medium, the

LOYOLA-VARGAS AND SANCHEZ DE JIMENEZ

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Plant Physiol. Vol. 76, 1984

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I 3 5 7 3 5 7 FIG. 3. Plantlet (A), root (B), and leaf (C) fresh weights after 20 d of plantlet growth. Each bar represents the average of 50 plantlets. Values are representative of four different experiments. 1, Water (control); 2, 20 mM NH4a; 3, 40 mM KNO3; 4, 10 mM ammonium citrate; 5, 40 mm KNO3 + 20 mm NH4a; 6, 10 mm glutamine; 7, 10 mm glutamic acid; 8, 10 mM urea. 1

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NITROOEN SOURCE FIG. 4. Effect of the nitrogen supply on the GDH-NADH (A) and GDH-NAD+ (B) activities of the maize callus during three successive subcultures. Each value is the average of three determinations. Other specifications as in Figure 2.

increase in fresh weight was maintained linearly during the subcultures. When only nitrate was present in the medium the callus grew well, although at a lower rate. On the other hand, when ammonium was the only nitrogen source, the calli grew more slowly and stopped at the fourth or fifth subculture. Additions of citrate to this medium resulted in good growth rates which were similar to those observed with nitrate alone. Organic nitrogen compounds were less efficient in supporting callus growth. Interestingly, on glutamate the callus showed a reproducible biphasic growth curve, suggesting a metabolic adaptation to this nitrogen source. Urea was the poorest nitrogen source even though the callus survived six subcultures (Fig. 2). The fresh weight of plant roots varied with the nitrogen source. Glutamate and urea resulted in lower growth values than the control plants (water). On the other hand, roots from plants grown in nitrate plus ammonium showed a 90% increase in fresh weight compared to the control (Fig. 3). The leaves of all plants showed a similar growth pattern.

Table I. Ratios of GDH in Different Nitrogen Sources A, GDH-NADH/GDH-NAD+ ratio in different nitrogen sources. B, Ratios expressed as fraction of MS value. MS value is taken as I. Subculture Nitrogen 2nd 4th 6th Source B A A B A B MS 5.3 1.0 8.0 1.0 11.8 1.0 Nitrate, 20 mm 5.4 1.0 6.2 0.8 5.0 0.4 5.1 1.0 Nitrate, 40 mm 3.7 0.5 6.9 0.6 5.2 1.0 15.0 1.9 12.4 Ammonium, 2 mM 1.0 Ammonium, 20 mM 6.6 1.2 16.0 2.0 9.1 0.8 Ammonium citrate, 10.2 1.9 13.7 1.7 11.0 0.9 10 mM 9.3 1.8 15.0 1.9 11.3 Glutamine, 5 mM 1.0 8.5 1.6 11.0 1.4 N.D. N.D. Urea, 10mM 4.7 0.9 5.1 0.6 Glutamate, 10 mM 4.6 0.4

GDH activity in the aminative (NADH) and deaminative (NAD+) pathways was measured in extracts from calli grown on the different nitrogen sources at the second, fourth, and sixth subcultures. Figure 4 shows values obtained for the second subculture. Ammonium and nitrate at 20 mm showed opposite effects; the former was inhibitory and the latter stimulatory for both activities. However, different behavior for the activities was observed in media containing NIV plus citrate, glutamine, or urea. GDH-NADH activity was not or only moderately affected by these compounds while GDH-NAD+ activity was strongly inhibited. To compare the effect of the nitrogen sources on the two pathways in the subcultures, ratios of all the activities were calculated and standardized by comparing them with those obtained in the MS medium (control) (Table I). These data indicate that the pathways do not respond in the same fashion with all the compounds tested. The GDH-NAD/NADH ratios tend to decrease in cultures with ammonium (even in the presence of citrate), as well as with glutamine or urea. On the other hand, glutamate, which is the substrate for the enzyme, and nitrate, tend to increase these ratios. In plantlets, roots had about 10 to 15 times more aminative activity and 6 to 10 times more deaminative activity per milli-

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roots, 20 mm ammonium chloride and, to a lesser extent, ammonium citrate and MS (nitrate plus ammonium) inhibited the deaminative reaction but barely influenced the aminative one (Figs. 5 and 6). In leaves, nitrate, ammonium citrate, glutamine, or urea significantly increased the deaminative path but did not affect the aminative one. Highest stimulation in leaves for both activities was observed when a balanced nitrogen source (NO3 plus NH4) was added to the plants.

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DISCUSSION As previously reported for other species-soybean (6), lucern 40' E and wheat (5), tobacco (3), and B. ternifolia (23)-maize cultures 600were not able to grow with ammonium as the sole nitrogen source, unless citrate was also added. This requirement is not 30 easily explained on the basis of the need for carbon skeletons to w a. z 400-I utilize ammonium, as has been suggested (3), since the tissues CO can grow even at higher ammonium concentrations in the ab0 20sence of extra carbon skeletons when nitrate is also present in E the medium (20) (Fig. 2). It seems, therefore, that this phenomct.a 200enon is more likely due to reducing power requirements (NADH) 10or to the transport requirements of the ion. Since callus growth improves after several transfers in ammonium (Fig. 2) in parallel with an increase of GDH-NADH activity (Table I), maize cul3 5 7 tures seem to overcome this problem in part by developing some 1 3 5 7 type of adaptation. NITROGEN SOUF Our results show that GDH-NADH and the GDH-NAD' levels do not have a similar response to different nitrogen comactivity of 4ADH of the nitrogen supply on the GDH-} FIG. 5. Effect root and leaf plantlets. Each value is the average of threw e determinations. pounds neither in calli nor in plant tissues. This is also supported by the behavior of the pathway observed through the growth Others specifications as in Figure 3. cycle (Fig. 1). These data could be explained either by the LEAVES presence of several isoenzymes with different rates for the amiROOTS native and deaminative reactions, or by the existence of different 200 H conformers of the same enzyme with preferential affinity for 40 NAD' or NADH, as it has recently been suggested for developing soybean seeds (1 1) and for the green alga Scenedesmus acutus Two other possible explanations, however, could be con(24). 0 sistent with these data: different protein levels and constant GDH protein and activity, or the presence of specific inhibitors or activators in the cultures. Further work is being carried out in CL 20 cx order to rule out any of these possibilities. The presence of this specialized system of enzymes, regulated 150> by the nitrogen metabolites available to the tissues, suggests that this pathway plays an important role in nitrogen metabolism, Ia both under normal and ammonium-stressed conditions. This role might depend on the state of differentiation of the tissue 100I (23). w z I0a. The larger amounts of GDH present in roots, as compared I CO, with the activity found in leaves (Fig. 5) suggest that under co normal conditions roots are a major site for ammonium assim501 E ilation in maize plantlets (18, 25). The increase in leaf fresh weight when the plants are supplemented with glutamine (Fig. c..a 3) is also consistent with this interpretation. E I

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of protein than leaves. This is consistent Nwith a previous GDH-NADH report using corn seedlings (7). In general terms i, GDH-NADH activity did not vary with the nitrogen source in roots; in leaves, it was very sensitive to the particular nitrogen souirce (Figs. 5 and 6). On the other hand, GDH-NAD' varied on bx)th tissues. Several interesting differences were observed v specific nitrogen sources on the aminative and deaminatiive pathways; in gram

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Acknowledgments-The biological material for this work was donated by the Internal Center for Wheat and Maize Improvement (CIMMYT), Mexico. The authors wish to thank Drs. Manuel Robert and Adolfo Garcia-SAinz for the critical

reading of this manuscript and Maricela Villarreal for skillful typing. LITERATURE

CITED

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LOYOLA-VARGAS AND SANCHEZ DE JIMENEZ

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